Recently, there have been several reports demonstrating improvements in the flow cytometric detection of intracellular cytokines. These advances, although significant, have not yielded techniques that have easily been translated into broad use. To address this issue, we have coupled a fixation and permeabilization method with the use of directly labelled monoclonal anti-cytokine antibodies, providing both improved signal and simpler staining. The kinetics of in situ cytokine production in both CD4 and CD8 cells are shown for IL-2, IL-4, IL-5 and IFN-gamma. Based on these data, 6 h was chosen for optimal detection of this combination of cytokines. We show the specificity of this technique by blocking cytokine staining using a molar excess of recombinant cytokine. Additionally, unlabelled anti-cytokine antibodies are demonstrated to block specific staining of labelled antibody, providing an objective means to place statistical markers. Using such controls, we routinely detected as few as 0.1% false positive cells, allowing the flow cytometric detection of IL-5, which is below the threshold of detection of published methods. To further prove the specificity of staining, we stained using two anti-IL-5 mAbs known to recognize different epitopes and demonstrate that the same cells stain with both antibodies. Without permeabilization we could detect a fraction of cells with low intensity staining for cytokine. This staining was further examined using differential two color staining for intracellular and extracellular cytokine, clearly demonstrating no cells staining exclusively for extracellular cytokine, confirming a lack of passive transfer of cytokine to nearby cells. We show that cytokine flow cytometry is useful in examining the increased IL-5 production characteristic of eosinophilic states and that IL-5 production is limited to the CD27 negative subpopulation. These data illustrate the unique capability of cytokine flow cytometry to correlate cytokine expression with cell surface phenotype without cell separation. In summary, using directly conjugated anti-cytokine antibodies, cytokine flow cytometry becomes a specific and versatile technique for the assessment of complex cytokine production phenotypes in fresh ex vivo T cell subpopulations.